1. Technical Field
The present invention relates to solid support materials used in diagnostic assays involving binding reactions, particularly antibody binding reactions, in which a fluorescent signal is used to detect the presence of bound analytes.
2. Background
There has been a rapid increase in the number and types of analyses employed to detect the presence or amount of analytes in samples of various origins in recent years. Most of these assays utilize the binding of one chemical or biological entity by another as the primary event by which an analyte is detected. However, this primary recognition event is not itself detectable by the person carrying out the assay since this binding interaction takes place on a submicroscopic level. Such assays therefore require the use of some detectable signal by which binding interactions themselves can be detected.
A number of detection techniques involve fluorescence. Solid-phase fluorescence immunoassays have been described in publications since 1965. However, techniques relying on fluorescence for detection of the binding interaction suffer from limitations that prevent them from achieving their theoretical limit of sensitivity. Two factors significantly affecting sensitivity are background fluorescence and light scattering by solid materials in contact with the reaction media, such as substrates to which reactants are attached or walls of containers in which measurements are made. These phenomena arise from the use of a light beam to excite the fluorescent signal material (fluorophore) being detected. The light used for excitation is of a shorter wavelength than the waveband of light emitted by the fluorescent material and accordingly does not normally interfere with the signal measurement itself. However, light in the excitation waveband may also cause fluorescence of the support material, thereby interfering with the sensitivity of detection when the emission waveband of the solid support overlaps the emission waveband of the fluorophore. Additionally, light scattering from ambient light of the wavelength being detected also interferes with detection of primary fluorescence by the signal material in some cases.
These problems are particularly acute when the solid is a polyamide. A number of studies show that light-stimulated endogenous fluorescent emissions and light reflection, from nylon-6,6 in particular, can coincide with the range of UV-visible wavelengths emitted from fluorophore-tagged analytes. Other materials utilized as solid supports also fluoresce and reflect light, but these materials do so to a lesser extent than polyamides. At the same time, recent developments in polyamide chemistry have created microporous membranes and other nylon materials having properties that are extremely useful in binding assays, such as controlled porosity, wettability, and enhanced surface charge.
Accordingly, there is a need for polyamide materials that will not exhibit background fluorescence or light reflectance to a degree that interferes with reliable detection of fluorescent emissions from fluorophore-tagged analytes. These materials also must retain the chemical and physical properties that give them their advantageous properties for use in analytical assays of the type described.
3. Relevant Literature
A number of review articles on fluorescence immunoassays have been published. See, for example, Jolley et al., Journal of Immunological Methods (1984) 67:21-35, and references cited therein. A study of a number of polymer substances for light absorbing and emission properties using UV-visible derivative spectroscopy is set forth in Allen, Polymer Photochemistry (1981) 1:43-55. A number of different dyes useful in carrying out the present invention are described in Lienhard et al., U.S. Pat. No. 4,527,994.